长链非编码RNA在移植静脉内膜增生中的作用及其力学生物学机制

The vein graft stenosis induced by neointimal hyperplasia is one of the major patency after coronary artery bypass grafting (CABG). The abnormal proliferation and migration of venous vascular smooth muscle cells (VSMCs) which is induced by mechanical stress are the major pathological basis. Preliminary study demonstrated that long noncoding RNA (lncRNA) which was modulated by mechanical stress (strain) played an important role in VSMC function. In this project, first, the lncRNA sequencing is applied to screen the different lncRNAs and SNPs between the graft vein and the control vein. And then the role of screened lncRNAs, especially which locus are nearby the cardiovascular genetic landmark or having binding sites with miRNA, is studied in order to illuminate the mechanisms of lncRNAs in regulation of proliferation and migration in venous VSMCs subjected to cyclic strain. Finally, the new screened lncRNA is knocked down in vein graft mice by RNAi in vivo or gene knockout mice, leading to identification of its role in vascular remodeling. With the employment of gene sequencing, this project study the network composed of lncRNA-microRNA-mRNA-protein and the specific lncRNA simultaneously . The project will achieve the deep insights into the role of lncRNA in the cyclic strain modulating VSMCs and mechanical stress-induced vascular remodeling. Further, the accomplishment of the project will provide and potential biomarkers and targets for vein graft stenosis therapy.

移植静脉内膜增生引起的再狭窄是影响冠状动脉旁路移植术预后的主要原因，机械应力引起移植静脉血管平滑肌细胞（VSMCs）异常增殖和迁移是其主要病理基础。前期研究发现应力（应变）调控的lncRNA可能影响VSMC功能。本项目拟筛选和鉴定小鼠移植静脉中差异表达的lncRNA及单核苷酸多态性；应用细胞张应变加载模型，着重研究空间位置上与已知心血管疾病重要致病基因相关或与miRNA分子有结合位点的lncRNA，如lincRNA-p21、 lncRNA Ang362、 H19 和MRAK048635等。阐明lncRNA在张应变调控静脉VSMC增殖和迁移中的分子机制；最后，回归动物模型检测lncRNA对内膜增生的影响。本项目从测序出发，将研究力学作用下lncRNA-miRNA-mRNA和靶蛋白的网络关系与特异lncRNA调控作用结合，深入研究lncRNA在调控细胞功能、参与应力诱导血管重建中的作用及机制。

静脉移植术后内膜增生引起血管再狭窄是手术失败的主要原因，机械应力引起移植静脉血管平滑肌细胞（vascular smooth muscle cells，VSMCs）异常增殖和迁移是其主要病理基础。目前尚无有效的防治手段，探索新的治疗方式和分子靶点十分必要。. 本项目通过对移植静脉和对照静脉进行全转录组测序和miRNA测序，筛选和鉴定移植静脉中差异表达非编码RNA，应用生物信息学方法结合细胞张应变加载模型和多种分子生物学方法，着重研究了几个长链非编码RNA在调控细胞功能、参与应力诱导血管重建中的作用及机制。取得了以下有意义的结果:1.首先完成了多个信号网络的构建，包括差异mRNA聚类和信号通路分析、差异 lncRNA 筛选和cis-lncRNA作用网络构建、ncRNA/circRNA-miRNA-mRNA ceRNA网络构建，找到了以关键长链非编码RNA（LncRNA MIAT, circRNA等）为核心的信号转导途径；2. 环状RNA是一类有共价闭合结构的长链RNA。我们发现CircRNA TET3通过吸附miR-351-5p调控VSMC迁移；环状RNA circUVRAG也可以影响VSMCs的粘附和迁移；并且深入研究环状RNA的成环机制，发现可变剪切因子NOVA1与pre-RNA circUVRAG共定位，可以影响circUVRAG的表达水平。另外，发现circDcbld1-miR-145-3p/Nrp1通路在大鼠颈动脉内膜损伤中的作用。在动物水平干扰circDcbld1能够减缓内膜损伤引起的内膜增生。3. VSMCs表型转化是其功能改变的起始事件。mir-29a-TET1通路在周期性张应变（应力）调控VSMC表型转化中发挥作用。4. 同时对于全转录组数据深入分析，预测227个基因可能发生了可变剪切现象。我们以VEGFA为研究切入点，发现高张应变条件下VEGFA发生可变剪切现象。并且这种可变剪切受到SRSF1转位的调控。这部分研究说明力学刺激确实可能影响基因的可变剪切过程。长链非编码RNA的可变剪切过程是将来研究的一个方向。.以上的结果表明lncRNA通过多种机制在张应变调控静脉VSMC功能中发挥作用，我们的发现丰富了移植静脉内膜增生的基本病理知识，几个关键分子可能成为预防和治疗静脉移植术后内膜增生的的新分子靶点。